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Proteomic Analysis of Polymeric Salivary Mucins

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Proteomic Analysis of Polymeric Salivary Mucins Proteomic analysis of polymeric salivary mucins: No evidence for MUC19 in human saliva Karine Rousseau, Sara Kirkham, Lindsay Johnson, Brian Fitzpatrick, Marj Howard, Emily J Adams, Duncan F Rogers, David Knight, Peter Clegg, David J Thornton To cite this version: Karine Rousseau, Sara Kirkham, Lindsay Johnson, Brian Fitzpatrick, Marj Howard, et al.. Proteomic analysis of polymeric salivary mucins: No evidence for MUC19 in human saliva. Biochemical Journal, Portland Press, 2008, 413 (3), pp.545-552. 10.1042/BJ20080260. hal-00478964 HAL Id: hal-00478964 https://hal.archives-ouvertes.fr/hal-00478964 Submitted on 30 Apr 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemical Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080260 Proteomic analysis of polymeric salivary mucins: No evidence for MUC19 in human saliva Karine Rousseau1, Sara Kirkham1, Lindsay Johnson1, Brian Fitzpatrick1, Marj Howard1, Emily J. Adams2, Duncan F. Rogers2, David Knight1, Peter Clegg1,3 & David J. Thornton1 1 Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK. 2Airway Disease, National Heart & Lung Institute, Imperial College London, London SW3 6LY, UK, 3University of Liverpool, Faculty of Veterinary Science, Leahurst, Neston, CH64 7TE, UK. Running head: Salivary mucins Corresponding author: David J. Thornton Wellcome Trust Centre for Cell-Matrix Research Faculty of Life Sciences The Michael Smith Building University of Manchester Manchester M13 9PT, UK. Tel: 0161 275 5647 Fax: 0161 275 1505 E-mail: [email protected] Key words: saliva, mucins, Muc19, Muc5b THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080260 Abbreviations: TR: tandem repeat; GdmCl: Guanidinium chloride; PAS: Periodic Acid Schiff's; PSM: Pig submaxillary mucin; BSM: Bovine submaxillary mucin; DTT: ditiothretiol; CsCl: Caesium chloride; MS-MS; Tandem mass spectrometry. Stage 2(a) POST-PRINT 1 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Biochemical Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080260 ABSTRACT MUC5B is the predominant polymeric mucin in human saliva (Thornton, Khan, Mehrotra, Howard, Veerman, Packer, and Sheehan (1999) Glycobiology 9, 293-302) where it contributes to oral cavity hydration and protection. More recently the gene for another putative polymeric mucin, MUC19, has been shown to be expressed in human salivary glands (Chen, Zhao, Kalaslavadi, Hamati, Nehrke, Le, Ann, and Wu, (2004) Am.J.Respir.Cell Mol.Biol. 30, 155-165). However, to date, the MUC19 mucin has not been isolated from human saliva. Our aim was therefore to purify and characterize the MUC19 glycoprotein from human saliva. Saliva was solubilised in 4M guanidinium chloride and the high-density mucins were purified by density gradient centrifugation. The presence of MUC19 was investigated using tandem mass spectrometry of tryptic peptides derived from this mucin preparation. Using this approach we found multiple MUC5B- derived tryptic peptides but were unable to detect any putative MUC19 peptides. These data suggest that MUC19 is not a major component in human saliva. In contrast, using the same experimental approach we identified Muc19 and Muc5b glycoproteins in horse saliva. Moreover, we also identified Muc19 from pig, cow and rat saliva; the saliva of cow and rat also contained Muc5b, although, due to the lack of pig Muc5b genomic sequence data, we were unable to identify Muc5b in pig saliva. Our data suggest that unlike human saliva which contains MUC5B, cow, horse and rat saliva is a heterogeneous mixture of Muc5b and Muc19. The functional consequence of these species differences remains to be elucidated. THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080260 Stage 2(a) POST-PRINT 2 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Biochemical Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080260 INTRODUCTION Saliva is a dilute, complex mixture of proteins, glycoproteins, lipids and ions secreted from the major and minor salivary glands [1-3]. Saliva has many diverse functions including; aiding mastication and speech, digestion of food and maintenance of oral health [4]. The major macromolecular components of normal, unstimulated saliva are the mucins. There are two-populations of salivary mucins; the high molecular weight polymeric, gel- forming mucin MUC5B and the lower molecular weight, non-polymeric mucin MUC7 [5- 7]. Two distinctive structural characteristics of these glycoproteins have important functional consequences [8]. First, the MUC5B and MUC7 mucin polypeptides, have a large central domain, with tandemly repeated sequences enriched in serine and/or threonine residues (the TR or mucin domain), which are the sites of extensive substitution with O- glycans. Second, upstream and downstream of the MUC5B mucin domain are regions of the polypeptide similar to von Willebrand Factor D domains, which are important for mucin polymer formation [8]. Thus, as a result of their extreme size and abundance of negatively charged O-glycans these glycoproteins play key roles in hydration and lubrication of the oral surfaces. Furthermore, mucins bind to, and sequester bacteria via their glycans and protein domains [9;10]. Until 2003, four gel-forming mucins had been reported, namely MUC6, MUC2, MUC5AC and MUC5B which are encoded by four consecutive genes on chromosome 11p15.5 [11]. These mucins are expressed in a tissue, and cell specific manner. For instance, in humans, MUC6 is expressed in mucous cells of submucosal glands in the stomach, MUC5B is mainly expressed by the mucous cells of the salivary glands and the submucosal glands in the airways, MUC5AC is expressed by the goblet cells of the airways and the stomach, and MUC2 is mainly expressed by the goblet cells in the intestine [12-17]. This tissue-specific distribution has been found to be relatively well conserved between species [18;19]. For example, in the mouse, Muc5b, along with Muc5ac, have been found to be expressed in the airways, Muc2 is mainly expressed in the intestine and Muc5ac and Muc6 are both found in the stomach [20-23]. However, differences in expression between mouse and human have also been reported. For example, in contrast to humans, no evidence of Muc5b (nor Muc2, Muc5ac or Muc6) expression has been reported in mouse saliva [22;23]. In 2003, MUC19 was reported as a fifth human gel-forming mucin. Although, neither the complete amino acid nor mRNA sequence have been published, MUC19 is predicted to have similar structural features to the other human gel-forming mucins [24]. THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080260 MUC19 gene expression has been reported in human airways and, in particular, in human salivary glands [24]. In the mouse, the complete sequence of the Muc19 gene has been described and it is strongly expressed in sublingual and submandibular salivary glands [24;25]. Furthermore, mRNA sequences from porcine and bovine submaxillary mucins show high sequence similarity to the mouse Muc19 sequence [26-28]. On the basis of these data, MUC19/Muc19 would be expected to be present in saliva from humans and other mammals. However, studies on human saliva have identified MUC5B as the predominant polymeric mucin [7;16], and the MUC19 glycoprotein has yet to be identified in human saliva, or for that matter, in the saliva from other mammals. Moreover, in other animals, it Stage 2(a) POST-PRINT 3 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Biochemical Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080260 is not known whether Muc5b is a major salivary mucin. Therefore, in this study, our first aim was to purify the polymeric mucins from human saliva in order to identify whether MUC19 was present. Subsequently, we analysed the saliva collected from horse, rat, pig and cow to identify if Muc5b and/or Muc19 were present. THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080260 Stage 2(a) POST-PRINT 4 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Biochemical Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20080260 EXPERIMENTAL METHODS Saliva collection: Unstimulated saliva was collected from 6 healthy human volunteers (between 20 and 35 of age) by spitting into 50 ml tubes that were kept on ice during the collection. Horse, cow and pig saliva were collected from excess secretions drooled while feeding. Necessary ethical approval was obtained for all the above animal sampling. Rats were anaesthetised with ketamine hydrochloride (100mg/kg) and xylazine (10mg/kg) by intraperitoneal injection and were injected intraperitoneally with the cholinergic agonist pilocarpine (25mg/kg) to stimulate salivary secretion. The anaesthetised rats were laid on their side and saliva dripping from the mouth was collected into plastic petri dishes. Experiments were carried out in accordance with the UK Home office guidelines for animal welfare based on the Animals (Scientific Procedures) Act 1986. Equal volumes of 8M guanidinium chloride (GdmCl) were added to each sample at the time of collection and up to 5 volumes of 4M GdmCl were subsequently added to solubilise the mucins. This step was performed by gentle agitation for at least 24 h at 4°C.
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